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EP0092004B1 - Dérivés de vitamine D3, procédés pour leur préparation, antigènes contenant ces composés utiles pour la préparation d'anticorps pour des tests immunochimiques et anticorps obtenus - Google Patents

Dérivés de vitamine D3, procédés pour leur préparation, antigènes contenant ces composés utiles pour la préparation d'anticorps pour des tests immunochimiques et anticorps obtenus Download PDF

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Publication number
EP0092004B1
EP0092004B1 EP19820302015 EP82302015A EP0092004B1 EP 0092004 B1 EP0092004 B1 EP 0092004B1 EP 19820302015 EP19820302015 EP 19820302015 EP 82302015 A EP82302015 A EP 82302015A EP 0092004 B1 EP0092004 B1 EP 0092004B1
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Prior art keywords
group
vitamin
preparation
hydrogen atom
antigen
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English (en)
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EP0092004A1 (fr
Inventor
Jun-Ichi Oshida
Osamu Nishikawa
Kenji Ishimaru
Hideki Tsuruta
Toru Takeshita
Itaru Yamamoto
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Teijin Ltd
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Teijin Ltd
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Priority to DE8282302015T priority patent/DE3267108D1/de
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07J—STEROIDS
    • C07J9/00—Normal steroids containing carbon, hydrogen, halogen or oxygen substituted in position 17 beta by a chain of more than two carbon atoms, e.g. cholane, cholestane, coprostane
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/82—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving vitamins or their receptors

Definitions

  • the present invention relates to novel vitamin D 3 derivatives, processes for the preparation thereof, and antigens comprising said derivatives for the preparation of antibodies for immunochemical assay and antibodies prepared therefrom.
  • the present invention relates to novel vitamin D 3 derivatives which have a carboxyl group or its ester group at the 22-position, processes for the preparation thereof, and antigens which comprise said vitamin D 3 derivative and an immunogenic carrier material for the preparation of antibodies for enzymeimmunoassay or radioimmunoassay and antibodies obtained by inoculating a host animal with said antigen for an enzymeimmunoassay or radioimmunoassay.
  • vitamin D 3 is metabolized in the kidney or liver into 25-hydroxyvitamin D 3 , 1a,25-dihydroxyvitamin D 3 , 24,25-dihydroxyvitamin D 3 , etc.
  • Such metabolites as 25-hydroxyvitamin D 3 , 1a,25-dihydroxyvitamin D 3 , etc. and such activated vitamin D 3 compounds as la-hydroxyvitamin D 3 , 1a,24-dihydroxyvitamin D 3 , etc. are used clinically as a remedy for osteoporosis, osteomalacia, etc. and their development is now taking place.
  • these remedies are clinically administered in very small dosages in general and a single dose is in the range of several hundreds ng to several dozens ⁇ g.
  • the pharmacological action of a remedy has the interrelationship with its concentration in the serum or tissue, and the determination of the concentration of the remedy is clinically very important in the case of human beings.
  • Japanese Patent Application Laid-Open No. 47653/80 discloses a radioimmunoassay wherein la-hydroxyvitamin D 3 3-hemisuccinate or 1a,25-dihydroxyvitamin D 3 3-hemisuccinate is used as a hapten.
  • Japanese Patent Application Laid-Open No. 87344/78 discloses a radioimmunoassay wherein 1a,25-dihydroxyvitamin D 3 25-hemisuccinate is used as a hapten.
  • antigens for use in the assays are those obtained by bonding a carrier protein to a hapten by means of a carboxyl group in the substituent group at the 25-position or the 3- position of said hapten.
  • antibody antihapten antibody
  • antigen for assay use recognizes the chemical structure on the hapten apart from the place of its binding site to the carrier protein.
  • vitamin D 3 derivatives which have a carboxyl group or its ester group at the 22-position and found that these vitamin D 3 derivatives are very useful as a hapten for an enzymeimmunoassay or radioimmunoassay which is preferably adopted for the determination of the concentration of an activated vitamin D 3 compound having a hydroxyl group at the 1 ⁇ -position, 24-position, or 25-position, thus achieving the present invention.
  • the present invention is concerned with:
  • antigens which are prepared by covalently bonding said vitamin D 3 derivative expressed by formula (I) through its carboxyl group or amino group to an immunogenic carrier material for the preparation of an antibody for immunochemical assay;
  • a novel vitamin D 3 derivative offered in the present invention is a compound expressed by the aforementioned formula (I) and has a characteristic chemical structure which has a carboxyl group or its ester group at the 22-position.
  • R 1 indicates a hydrgen atom or an alkyl group with 1 to 6 carbon atoms having a carboxyl group or amino group.
  • alkyl groups with 1 to 6 carbon atoms having a carboxyl group or amino group there may be exemplified alkyl groups with 1 to 6 carbon atoms having a carboxyl group such as carboxymethyl group, 2-carboxylethyl group, 3-carboxypropyl group, 4-carboxybutyl group, 5-carboxypentyl group, 6-carboxyhexyl group, etc.
  • alkyl groups with 1 to 6 carbon atoms having an amino group such as aminomethyl group, 2-aminoethyl group, 3-aminopropyl group, 4-aminobutyl group, 5-aminopentyl group, 6-aminohexyl group, etc.
  • a hydrogen atom is especially preferable as R 1 .
  • R 2 , R 3 , and R 4 independently indicate a hydrogen atom or hydroxyl group.
  • These vitamin D 3 derivatives whose R 2 and R 3 are both hydroxyl groups are especially preferable as haptens for assaying activated vitamins D 3 such as 24, 25-dihydroxyvitamin D 3 .
  • vitamin D 3 derivatives expressed by the aforementioned formula (I) include the following:
  • vitamin D 3 derivatives of the present invention are prepared as follows. Their preparation is carried out by subjecting a cholesta-5,7-diene derivative expressed by the following formula (II) wherein definitions of R' i , R' 2 , R' 3 , R' 4 , and R s are the same as those given earlier, to the thermal isomerization after ultraviolet irradiation in an inert organic medium, followed by deprotection, if necessary.
  • formula (II) wherein definitions of R' i , R' 2 , R' 3 , R' 4 , and R s are the same as those given earlier
  • the material compound, or said cholesta-5,7-diene derivative expressed by formula (II), can be prepared from its corresponding cholest-5-ene derivative by allylic bromination followed by dehydrobromination.
  • R' indicates a hydrogen atom, alkyl group with 1 to 6 carbon atoms, alkyl group with 1 to 6 carbon atoms having a carboxyl group or amino group, or alkyl group with 1 to 6 carbon atoms having a protected carboxyl group or protected amino group.
  • alkyl groups with 1 to 6 carbon atoms there may be exemplifed a methyl group, ethyl group, propyl group, butyl group, t-butyl group, pentyl group, hexyl group, etc.
  • alkyl groups with 1 to 6 carbon atoms having a carboxyl group or amino group there may be mentioned those given as R, in the aforementioned formula (I).
  • alkyl groups with 1 to 6 carbon atoms having a protected carboxyl group or protected amino group the following ones may be exemplified.
  • alkyl groups with 1 to 6 carbon atoms having a carboxyl group cited as examples of R, of the aforementioned formula (I), wherein said carboxyl group is a group which forms an ester bond may be mentioned including, for instance, a methoxycarbonylmethyl group, ethoxycarbonylmethyl group, butoxycarbonylmethyl group, 2-methoxycarbonylethyl group, 2- ethoxycarbonylethyl group, 2-butoxycarbonylethyl group, etc.
  • alkyl groups with 1 to 6 carbon atoms having a protected amino group those alkyl groups with 1 to 6 carbon atoms having an amino group cited as examples of R, of the aforementioned formula (I), wherein said amino group is a group which forms an amid bond, may be mentioned including, for instance, an acetylaminomethyl group, 2-acetylaminoethyl group, 4-acetylaminobutyl group, etc.
  • R' 2 , R' 3 , and R' 4 independently indicate a hydrogen atom, hydroxyl group, or protected hydroxyl group and R 5 represents a hydrogen atom or protecting group.
  • protecting groups in the case where R' 2 , R' 3 , and R' 4 are protected hydroxyl groups or where R 5 is a protecting group, there may be mentioned, for instance, aliphatic or aromatic carboxylic acid residues with 1 to 12 carbon atoms such as an acetyl group, propanoyl group, butanoyl group, pivaloyl group, pentanoyl group, cyclohexanoyl group, chloroacetyl group, bromoacetyl group, benzoyl group, p-bromobenzoyl group, p-nitrobenzoyl group, ethylbenzoyl group, toluyl group, etc.
  • cholesta-5,7-diene derivatives expressed by the aforementioned formula (II) are thermally isomerized after ultraviolet irradiation in an inert organic medium.
  • ultraviolet rays to be used for ultraviolet irradiation those having wavelengths of about 200 to 360 nm, preferably those having wavelengths of 260 to 310 nm, are used.
  • the ultraviolet irradiation reaction is carried out in an inert organic medium.
  • inert organic mediums hydrocarbon or halogenated hydrocarbon such as hexane, heptane, benzene, toluene, xylene, chlorobenzene, and carbontetrachloride; ethers such as diethyl ether, tetrahydrofuran, and dioxane; or alcohols such as methanol, ethanol, and propanol are used as desirable ones.
  • reaction temperature has not much significance; however, the reaction is usually carried out at -20°C to 120°C, especially in the range of -10°C to 50°C.
  • the isomerization reaction is conducted at 20 to 120°C, preferably at 40 to 100°C.
  • This isomerization reaction can be satisfactorily conducted in the inert organic medium in which said ultraviolet irradiation has been carried out.
  • previtamin D 3 derivative which is formed as C-C bond is being cleaved at the 9- and 10-positions of cholesta-5,7-diene derivative, gradually isomerizes into vitamin D 3 derivative in the same reaction system coincidently.
  • the isomerization reaction by thermal energy proposed in the present invention does not necessarily mean the heating of the reaction system as clearly understood from what is mentioned above.
  • Said vitamin D 3 derivative expressed by formula (I) or its derivative having protected hydroxyl group, protected carboxyl group, or protected amino group is thus prepared.
  • said isomerization reaction must be followed by the removal of the protecting groups.
  • This deprotection reaction is itself a well known reaction and may be carried out, for instance, when the protecting group forms an acyl group, by treating with an alkaline solution in lower aliphatic alcohol such as methanol and ethanol or with metal hydride such as LiAIH 4 in ether.
  • the reaction temperature may be in the range of -10°C to 50°C.
  • the protecting group is bonded to an oxygen atom of a hydroxyl group to form an ether group, it can be easily removed by reductive treatment or by treatment with acid or alkali.
  • the vitamin D 3 derivative in the case where R, of said vitamin D 3 derivative expressed by formula (I) is an alkyl group with 1 to 6 carbon atoms having a carboxyl group or amino group, the vitamin D 3 derivative can also be obtained by the following procedure. It can be prepared by subjecting vitamin D 3 derivative expressed by the aformentioned formula (I) in which R, is a hydrogen atom and hydroxy acid with 1 to 6 carbon atoms having a carboxyl group such as glycolic acid, lactic acid, p-hydroxypropionic acid, etc. or an amine compound with 1 to 6 carbon atoms having an amino group such as 2-hydroxyethylamine, 3-hydroxy- butylamine, etc. to the esterification reaction. The isolation and the purification of the obtained vitamin D 3 derivative is accomplished by column chromatography, thin-layer chromatography, high-pressure liquid chromatography, recrystallization, etc.
  • Vitamin D 3 derivatives expressed by formula (I) offered by the present invention are novel compounds which find no mention in any literature and have a characteristic chemical structure of having a carboxyl group or its ester group at the 22-position. Such vitamin D 3 derivatives are very useful as hapten for enzymeimmunoassay or radioimmunoassay which is desirable for the determination of activated vitamin D 3 compound having a hydroxyl group at the 1a-, 24-, or 25-position.
  • said vitamin D 3 derivative expressed by formula (I) is used as hapten for enzymeimmunoassay or radioimmunoassay, said hapten is covalently bonded to an immunogenic carrier material to form an antigen for the preparation of an antihapten antibody.
  • An antibody prepared from the antigen is offered as one of the reagents which make up the assay kit for enzymeimmunoassay or radioimmunoassay.
  • the present invention offers an antigen which is formed by covalently bonding said vitamin D 3 derivative expressed by formula (I) to an immunogenic carrier material through the carboxyl group or amino group of the vitamin D 3 derivative for the preparation of an antibody for immunochemical assay, and an antibody which is induced by inoculating a host animal with said antigen for immunochemical assay.
  • proteins As immunogenic carrier materials for obtaining an antigen by covalent bonding to a vitamin D 3 derivative, there may be mentioned proteins, polypeptide, glycoprotein, etc.
  • suitable proteins include bovine serum albumin (BSA), human serum albumin (HSA), human gamma globulin, methylated bovine serum albumin, rabbit serum albumin, and bovine gamma globulin.
  • polypeptides polylysine, poly-L-lysine-polyglutamic acid copolymer, etc. may be cited as examples.
  • glycoproteins lipopolysaccharide, ficoll, keyholelymphet, hemocyanin, etc. may be mentioned. Of them, proteins are preferred and bovine serum albumin and human serum albumin are especially preferred.
  • the covalent coupling of said vitamin D 3 derivative expressed by formula (I) to an immunogenic carrier material is carried out directly or indirectly through a carboxyl group or amino group at the 22-position of the vitamin D 3 derivative and a functional group of the immunogenic carrier material.
  • functional groups of the immunogenic carrier material an amino group of lysine, carboxyl group or amino group of aspartic acid or glutamic acid, phenolic hydroxyl group in a tyrosine residue, and mercapto group of cysteine, for instance, may be mentioned.
  • methods of covalent bonding there may be mentioned a method in which N-hydroxysuccinimide or alkylchloroformate is reacted with a carboxyl group or amino group of the vitamin D 3 derivative to activate the carboxyl group or amino group of the vitamin D 3 derivative, followed by covalent bond formation; a method in which a condensing agent such as 1-ethyl-3-(3-dimethylaminopropyl)-carboxyimide hydrochloride, dicyclohexylcarbodiimide, N-ethyl-5-phenylisoxazolium-3'-sulfonate, etc. is used; or a method in which both of these methods are used in combination.
  • Especially preferably method is one in which an activating agent such as N-hydroxysuccinimide, etc. and a condensing agent such as dicyclohexylcarbodiimide are used together.
  • An example of preferred embodiments is one in which a solution of, for instance, 22-carboxy-24,25-dihydroxycholecalciferol, N-hydroxysuccinimide, and dicyclohexylcarbodiimide in tetrahydrofuran is left standing at room temperature for some dozen hours to form N-hydroxysuccinimide ester of 22-carboxy-24,25-dihydroxycholecalciferol, which is then reacted with human serum albumin in a buffer solution of phosphoric acid.
  • antigen for the preparation of an antibody for immunochemical assay is inoculated into a host animal to induce the formation of an antibody.
  • warm-blooded animals such as rabbits, rats, cows, sheep, etc.
  • the inoculation of the antigen into a host animal is practised by extrabuccal administration, for instance, by means of hypodermic or intradermal injection.
  • the inoculation of the antigen is usually conducted with the combined use of Freund's complete adjuvent.
  • the inoculation is conducted by injecting 200 ⁇ g of the same antigen hypodermically or intradermally at an interval of four weeks. During the period of inoculation, the antibody concentration of the blood is determined every ten days and the total blood is withdrawn when the highest titer is obtained.
  • the serum separated from thus obtained blood may be used as antigen without any treatment, however, it is preferable to adjust the antibody concentration by obtaining IgG fractions by means of ammonium sulfate fractionation or gel filtration.
  • respective antibodies thus obtained can recognize activated vitamin D 3 having a hydroxyl group at the 1a-,24-, or 25-position such as compounds of la-hydroxy vitamin D 3 , 25-hydroxy vitamin D 3 , 1a, 25-dihydroxy vitamin D 3 , and 24,25-dihydroxy vitamin D 3 , they are useful as antibodies for enzymeimmunoassay or radioimmunoassay.
  • enzyme-labelled antigens cited herein those which can be obtained by covalently coupling said vitamin D 3 derivative expressed by formula (I) to such enzymes as ⁇ -D-galactosidase, alkaline phosphatase, glucose oxidase, lipase, peroxidase, etc., preferably such enzymes as ⁇ -D-galactosidase and alkaline phosphatase may be mentioned.
  • the antibodies of the present invention are used together with 24,25-dihydroxy vitamin D 3 , 25-hydroxy vitamin D 3 , la-hydroxy vitamin D 3 , and 1a-25-dihydroxyvitamin D 3 which are radiolabelled with 3 H (tritium) or 14 C as one of the reagents which make up the assay kit.
  • said antibodies can be used together with 125 l-Protein A, which is labelled with 125 l, as reagents for radioimmunoassay which is called Sandwich method.
  • 1,3-Dibromo-5,5-dimethylhydantoin (137.7 mg) was added to a refluxing solution of 22-methoxycarbonyl-3 ⁇ ,24-diacetoxycholest-5-en-25-ol (450 mg) in carbon tetrachloride (10 ml), and the mixture was stirred for 15 minutes under a nitrogen atmosphere with an infrared lamp irradiation. The mixture was then cooled and filtered. Evaporation of the filtrate gave a crude product. A solution of the crude product in xylene (12 ml) was added dropwise to a refluxing solution of xylene (7.5 ml) and s-collidine (2.5 ml) and the mixture was refluxed for 20 minutes.
  • the resulting dicyclohexylurea precipitate was eliminated by centrifugation (10000 g x 10 min).
  • An anti-serum usable for radioimmunoassay or enzymeimmunoassay was obtained in this way in about 4 months after the immunization was started.
  • a calibration curve was drawn by determining the respective ratios ( 125 l-radioactivity (B/Bo %) of y-counts between the two kinds of solutions, one without addition of la,25-dihydroxy vitamin D 3 and the other with addition of 1 ⁇ ;25-dihydroxy vitamin D 3 , following the above procedure with the concentration 1a,25-dihydroxyvitamin D 3 changed variously.

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Claims (15)

1. Dérivé de vitamine D3, exprimé par la formule suivante (I)
Figure imgb0014
dans laquelle R1 est un atome d'hydrogène ou un groupe alkyle ayant de 1 à 6 atomes de carbone, possédant un groupe carboxyle ou un groupe amino; R2, R3 et R4 indiquent, indépendamment les uns des autres, un atome d'hydrogène ou un groupe hydroxyle.
2. Dérivé de vitamine D3 selon la revendication 1, où R1, dans la formule (1) mentionée ci-dessus, est un atome d'hydrogène.
3. Dérivé de vitamine D3 selon la revendication 1, où R2 et R3, dans ladite formule (I), sont tous les deux un groupe hydroxyle.
4. Procédé pour la préparation d'un dérivé de vitamine D3, exprimé par la formule (I) ci-après
Figure imgb0015
dans laquelle les définitions de R1, R2, R3 et R4 sont les mêmes que ce ci-dessus, procédé caractérisé en ce qu'il consiste à soumettre un dérivé de cholesta-5,7-diène, exprimé par la formule (II) ci-après
Figure imgb0016
dans laquelle R'1 est un atome d'hydrogène, un groupe alkyle ayant de 1 à 6 atomes de carbone, un groupe alkyle ayant de 1 à 6 atomes de carbone possédant un groupe carboxyle ou un groupe amino, ou bien un groupe alkyle ayant de 1 à 6 atomes de carbone possédant un groupe carboxyle protégé ou un groupe amino protégé; R'2, R'3 et R'4 indiquent, indépendamment les uns des autres, chacun un atome d'hydrogène, un groupe hydroxyle ou un groupe hydroxyle protégé, et R5 est un atome d'hydrogène ou un groupe protecteur; à une isomérisation thermique après irradiation aux ultraviolets dans un milieu organique inerte, suivie si nécessaire d'une déprotection.
5. Procédé de préparation selon la revendication 4, dans lequel R'1 dans la formule (II) mentionnée ci-dessus, est un groupe alkyle ayant de 1 à 6 atomes de carbone.
6. Procédé de préparation selon la revendication 4, dans lequel, dans la formule (II) ci-dessus, R'2, R'2 et R'4 sont des groupes hydroxyles protégés et R5 est un groupe protecteur.
7. Antigène, préparé en fixant par liaison covalente un dérivé de vitamine D3 exprimé par la formule suivante (1)
Figure imgb0017
dans laquelle les définitions de R1, R2, R3 et R4 sont les mêmes que ce qui a été défini ci-dessus, par l'intermédiaire de son groupe carboxyle ou de son groupe amino, à un produit immunogène à titre de support pour la préparation d'un anticorps pour analyses immunochimiques.
8. Antigène selon la revendication 7, dans lequel le support immunogène est une protéine.
9. Antigène selon la revendication 7, dans lequel le support immunogène est la sérum-albumine bovine (BSA) ou la sérum-albumine humaine (HSA).
10. Antigène selon l'une quelconque des revendications 7 à 9, dans lequel R1, dans la formule (1) mentionnée ci-dessus, est un atome d'hydrogène.
11. Anticorps dont la formation résulte de l'injection, à un animal hôte, dudit antigène préparé en fixant par liaison covalente le dérivé de vitamine D3, exprimé par la formule (I) ci-après:
Figure imgb0018
dans laquelle les définitions de R1, R2, R3 et R4 sont les mêmes que ci-dessus, par l'intermédiaire de son groupe carboxyle ou de son groupe amino, à un support immunogène, pour une utilisation en analyse :mmunochimique.
12. Anticorps selon la revendication 11, dans lequel le support immunogène est une protéine.
13. Anticorps selon la revendication 11, dans lequel le support immunogène est la sérum-albumine bovine (BSA) ou le sérum-albumine humaine (HSA).
14. Anticorps selon l'une quelconqe des revendications 11 à 13, dans lequel R1, dans la formule (I) mentionnée ci-dessus, est un atome d'hydrogène.
15. Anticorps selon l'une quelconque des revendications 11 à 14, où l'animal hôte est un lapin.
EP19820302015 1982-04-20 1982-04-20 Dérivés de vitamine D3, procédés pour leur préparation, antigènes contenant ces composés utiles pour la préparation d'anticorps pour des tests immunochimiques et anticorps obtenus Expired EP0092004B1 (fr)

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Application Number Priority Date Filing Date Title
EP19820302015 EP0092004B1 (fr) 1982-04-20 1982-04-20 Dérivés de vitamine D3, procédés pour leur préparation, antigènes contenant ces composés utiles pour la préparation d'anticorps pour des tests immunochimiques et anticorps obtenus
DE8282302015T DE3267108D1 (en) 1982-04-20 1982-04-20 Novel vitamin d3 derivatives, processes for preparation thereof, and antigens comprising said derivatives to be used for preparation of antibodies for immunochemical assay and antibodies prepared therefrom

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EP19820302015 EP0092004B1 (fr) 1982-04-20 1982-04-20 Dérivés de vitamine D3, procédés pour leur préparation, antigènes contenant ces composés utiles pour la préparation d'anticorps pour des tests immunochimiques et anticorps obtenus

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Cited By (1)

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US9746483B2 (en) 2009-10-27 2017-08-29 Diasource Immunoassays S.A. Process for the production of a hybridoma and antibody obtained therefrom, able to recognize more than one vitamin D metabolite

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FR2631025B1 (fr) * 1988-05-04 1991-04-12 Ire Medgenix Sa Nouveaux derives de la vitamine d3 et application aux dosages des metabolites de la vitamine d3
EP0813423B1 (fr) * 1995-01-23 2002-07-03 Xenotech Incorporated Procede visant a inhiber l'osteolyse et les metastases
GB9623569D0 (en) * 1996-11-13 1997-01-08 Immunodiagnostic Systems Ltd Vitamin D immumoassay systems
WO2011050833A1 (fr) * 2009-10-27 2011-05-05 Diasource Immunoassays S.A. Procédé de production d'hybridomes et d'anticorps monoclonaux susceptibles de reconnaître des métabolites de la vitamine d
US8785603B2 (en) 2011-05-20 2014-07-22 Siemens Healthcare Diagnostics Inc. Antibodies to 25-hydroxyvitamin D2 and D3 and uses thereof
CN109575123B (zh) * 2018-11-08 2020-12-15 中国农业大学 一种氟乙酰胺半抗原及单克隆抗体的制备方法与应用
CN112759642B (zh) * 2019-11-05 2022-12-23 广东菲鹏生物有限公司 完全抗原及其制备方法、应用和产品
CN113391076B (zh) * 2021-07-20 2023-04-07 深圳健丰源科技有限公司 一种25-羟基维生素d的免疫检测方法及其应用

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GB1589921A (en) * 1977-01-07 1981-05-20 Hoffmann La Roche Radioimmunoassay for the determination of cholecalciferol
GB1592170A (en) * 1977-01-07 1981-07-01 Hoffmann La Roche 1a-hydroxycholecalciferol-25-hydroxy esters and their use in radioimmunoassay

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US9746483B2 (en) 2009-10-27 2017-08-29 Diasource Immunoassays S.A. Process for the production of a hybridoma and antibody obtained therefrom, able to recognize more than one vitamin D metabolite

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